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Effects of Stachybotrys chartarum on Surfactant Convertase Activity in Juvenile Mice
Toxicology and Applied Pharmacology 172, 21–28 (2001) doi:10.1006/taap.2001.9127, available online at http://www.idealibrary.com on
Effects of Stachybotrys chartarum on Surfactant Convertase Activity in Juvenile Mice C. D. Mason,* T. G. Rand,* M. Oulton,† J. MacDonald,† and M. Anthes† *Department of Biology, Saint Mary’s University, Halifax, Nova Scotia, Canada; and †Department of Physiology and Obstetrics and Gynecology, Dalhousie University, Halifax, Nova Scotia, Canada August 2, 2000; accepted January 9, 2001
nificantly elevated compared to those from other treatment groups, while LB phospholipid concentrations were significantly increased compared to saline and untreated control animal groups. These results show that S. chartarum spores significantly alter convertase activity in both the H and LB surfactant fractions in juvenile mice and that these changes can be related to changes in protein and phospholipid concentrations in alveolar lavage fractions. As surfactant promotes lung stability by reducing the surface tension of the air–alveolar interface, these results further support our position that inhalation exposure to S. chartarum spores in exposed individuals may lead to altered surfactant metabolism, and possibly to lung dysfunction through diminished alveolar surfactant surface tension attributes, and lung stability. © 2001 Academic Press Key Words: alveolar type II cells; convertase; fungal conidia; satratoxins; intratracheal instillation; surfactant.
Effects of Stachybotrys chartarum on Surfactant Convertase Activity in Juvenile Mice. Mason, C. D., Rand, T. G., Oulton, M., MacDonald, J., and Anthes, M. (2001). Toxicol. Appl. Pharmacol. 172, 21–28. We have shown recently that alveolar type II cells are sensitive to exposure to Stachybotrys chartarum spores, both in vitro and in an in vivo juvenile mouse model. In mice, this sensitivity is manifest in part as a significant increase in the newly secreted, biologically active, heavy aggregate form of alveolar surfactant (H) and the accumulation of the lighter, “metabolically used”, biologically inactive alveolar surfactant forms (L vivo) in the interalveolar space. Conversion of the heavy, surface-active alveolar surfactant to the light metabolically used, nonsurface active forms is believed to involve the activity of an enzyme, namely convertase, which is thought to be derived from lamellar bodies (LB) in alveolar type II cells. The purpose of this study was to evaluate the effects of S. chartarum spores on mouse H and LB convertase activity by measuring their rates of conversion to L vivo using the in vitro surface area cycling technique. It was determined whether there were concurrent changes in the protein and phospholipid concentrations of the raw bronchoalveolar lavage fluid (RL) and LB fractions that could be correlated with changes in convertase activity. Conversions of H to L vivo in untreated control mice and saline-, isosatratoxin F-, and Cladosporium cladosporioides-exposed mice were not significantly different (p > 0.05). However, conversion from H to L vivo in the mice exposed to S. chartarum spores was significantly higher than all other treatment groups (p < 0.001). LB to L vivo conversions in untreated and salineexposed mice were not significantly different, although they were significantly higher than the H to L vivo conversions in these two animal treatment groups (p < 0.005), which supports the position that LB is a source of convertase activity in animals. LB to L vivo conversion from C. cladosporioides-, isosatrotoxin F-, and S. chartarum-exposed mice were all significantly depressed (p < 0.003) compared to the LB to L vivo conversion values obtained from untreated and saline-exposed mice. Protein concentrations in RL, H, L vivo, and LB from mice exposed to S. chartarum spores were significantly elevated compared to those from the other treatment groups (p < 0.001). Protein concentration in H isolated from C. cladosporioides-exposed mice was also significantly elevated above untreated and saline control animal levels. Phospholipid concentrations in H isolated from S. chartarum-exposed mice were sig-
Stachybotrys chartarum (atra) is an important toxigenic fungus often associated with chronically wet cellulose-based building materials. Occupational inhalation exposure to the spores, mycelial fragments, and dust, all containing toxins of this species, has been implicated as the cause of a variety of health problems. Symptoms include mucous membrane irritation, respiratory and nervous system disorders, and immune system dysfunction (Croft et al., 1986; Hodgson et al., 1998; Johanning et al., 1993, 1996). Despite the variety of symptoms associated with inhalation exposure to S. chartarum, there have been only a few in vivo animal studies evaluating the impact of spores of this species on lung tissue. Studies in inhalation toxicology have demonstrated the usefulness of bronchoalveolar lavage (BAL) fluid analysis for evaluating pulmonary dysfunction and damage (Anonymous, 1989; Das et al., 1997; Lopez and Yong, 1986; Oulton et al., 1993, 1994; Veldhuizen et al., 1997). The methods used are considered highly sensitive, quantifiable, and reproducible, with numerous advantages over routine histopathological evaluation of lung (Lopez and Yong, 1986). Previous work in our laboratory has led to the development of a relatively simple but useful method for evaluating pulmonary cell damage and lung dysfunction independent of the concurrent inflammatory lung 21
0041-008X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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response caused by exposure to toxic particulates (Oulton et al., 1991, 1993; Mason et al., 1998; Sumarah et al., 1999). This method is based on changes in the distribution or composition of alveolar surfactant subfractions, which are easily sampled and evaluated from the BAL fluid (Oulton et al., 1991, 1993). Mason et al. (1998) and Sumarah et al. (1999) used this methodology to show that alveolar type II cells, which comprise about 60% of the alveolar lining epithelium (Burkitt et al., 1993), are highly sensitive to exposure to S. chartarum spores. This sensitivity is manifest by significant alterations in the normal metabolic processing of alveolar surfactant in experimentally exposed mice. Mason et al. (1998) demonstrated that this sensitivity involves a significant increase in the newly secreted, biologically active, heavy aggregate form of alveolar surfactant and the subsequent accumulation of the lighter, “metabolically used”, biologically inactive alveolar surfactant forms in the interalveolar space. Sumarah et al. (1999) have shown that S. chartarum spore and toxin exposure induces significant changes in the phospholipid composition in the in vivo mouse model of lung injury, including depression of disaturated phosphatidylcholine (DSPC), the major surfactant phospholipid, which we believe may lead to lung dysfunction through altered pulmonary surfactant surface tension attributes. MaCrae et al. (2001) have revealed that the pulmonary surfactant compositional changes in S. chartarum spore-exposed mice may be due to molecular and biochemical alterations of the phospholipid components of pulmonary surfactant, resulting in a significant elevation in lysophospholipid content, which is a recognized accompaniment of alveolar type II cell damage (Scott et al., 1986, 1988). Mason et al. (1998) suggested that the accumulation of the metabolically used, biologically inactive alveolar surfactant forms could be the result of a disruption in the conversion of heavy surfactant to light or in the mechanisms of clearance of the converted surfactant from the alveolar space. Conversion of the heavy to light forms of pulmonary surfactant requires the activity of a convertase enzyme, a tentatively identified 76-kDa serine protease (Gross and Schultz, 1990, 1992). This enzyme has been identified in many species, including mice (Dhand et al., 1994). Studies by Dhand et al. (1996) and Oulton et al. (1999) suggest that lamellar bodies (LB), the intracellular storage form of surfactant, may be the source of this activity. Gross and Narine (1989) developed a method to study convertase activity in vitro. The method involves an end-over-end rotation of surfactant samples, which mimics the changing surface area of the lungs during respiration. This technique has been used by investigators to characterize the activity of the convertase enzyme and alveolar surfactant metabolism (Gross and Schultz, 1992; Oulton et al., 1999; Ueda et al., 1994) and to assess its activity in lamellar bodies (Oulton et al., 1999; Dhand et al., 1996). This technique has also been used to give insight into the effects of lung injury on intraalveolar metabolism of surfactant (Gross, 1991; Higuchi et al., 1992; Ueda et al., 1994; Veldhuizen et al., 1997).
The purpose of the present study was to use the in vitro cycling technique to determine whether S. chartarum spore exposure affects the conversion of heavy to light alveolar surfactant subfractions and/or convertase activity in lamellar bodies and contributes to the accumulation of the light form isolated from lung tissues of juvenile mice. It was also to determine whether there were concurrent changes in the protein and phospholipid concentrations of the raw bronchoalveolar lavage fluid (RL) and LB fractions that could be correlated with changes in convertase activity. Based on results of our previous studies, we hypothesize that the accumulation of the metabolically used, biologically inactive alveolar surfactant forms in mouse lung is the result of a disruption in the conversion of heavy surfactant to the lighter, biologically inactive alveolar surfactant forms. METHODS Preparation of treatments. The S. chartarum isolate (spore dimensions 11.6 ⫾ 1.2 m long ⫻ 5.7 ⫾ 0.92 m wide; n ⫽ 240) used was recovered from a Hawaiian hotel and has been deposited in the Department of Agriculture Canada Culture Collection (DAOM 225489). The Cladosporium cladosporioides isolate (spore dimensions 6.3 ⫾ 0.9 m long ⫻ 2.5 ⫾ 0.51 m wide; n ⫽ 200) was used as a negative control and was recovered from an outdoor sampling site in Nova Scotia (T.G.R.). Both isolates were cultured on 2% MEA, at 25°C for 2 to 3 weeks. The spores were harvested by flooding the cultures with sterile 0.9% NaCl followed by gentle agitation using a heatsterilized Pasteur pipette, and then collected and concentrated by centrifugation at 5000 rpm for 3 min in 1.5-ml Eppendorf centrifuge tubes. The spores were then suspended in 0.9% NaCl at a concentration of 1.4 ⫻ 10 6 conidia/ml, as per methods previously described in Mason et al. (1998). The suspensions were each used for intratracheal instillations within 2 h of isolation. Isosatratoxin F was employed as a positive control to determine whether the observed effects were due to exposure to trichothecene toxin sequestered in S. chartarum spores. It was isolated from an S. chartarum strain by Dr. Bruce Jarvis of the University of Maryland (College Park, MD). The toxin was dissolved in absolute methanol and then mixed to a concentration of 10 g/ml in 0.9% NaCl. Animals. Random-bred, pathogen-free, Carworth Farms White (CFW), Swiss Webster, male mice, 3– 4 weeks old were used in these experiments. The mice were housed according to the standards of the Canadian Council for Animal Care (CCAC, 1993). The mice were given food and water ad libitum. The animals were acclimatized for 1 week prior to usage. Intratracheal instillations. Control (untreated) and treatment animals (S. chartarum, C. cladosporioides, isosatratoxin F, and saline) were separated into groups of four or five mice each. A minimum of five groups was reserved for each treatment. The mice were anesthetized for instillation using an intramuscular injection of a mixture of ketamine (Ketaleen) and xylazine (Rompun) as previously described in Mason et al. (1998). The intratracheal instillation procedure used was a modification of the method described by Brain et al. (1976). It is described in detail in Mason et al. (1998). Each mouse, with the exception of untreated control mice, was instilled with either 50 l of S. chartarum or C. cladosporioides (1.4 ⫻ 10 6 conidia/ml), 10 g/ml of isosatratoxin F, or 0.9% NaCl. The mice were put back into their cages immediately after instillation and allowed to recover for 24 h. During recovery, the mice were continuously monitored for signs of sickness or distress as outlined in CCAC guidelines (CCAC, 1993). If the animals showed signs of distress, they were immediately euthanized using a sodium pentobarbitol (65 mg/ml) overdose and excluded from the study. Isolation of heavy aggregate alveolar surfactant. The exposed mice were killed after 24 h using a 300 l ip injection of 65 mg/ml sodium pentobarbitol. This
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Stachybotrys chartarum AND ALVEOLAR SURFACTANT time was chosen because results of our previous studies (see Mason et al., 1998; Sumarah et al., 1999) indicated that the most significant changes in surfactant composition and homeostasis were manifest between 24 and 48 h postinoculation. Mouse lungs were lavaged with 0.9% NaCl in 4 ⫻ 0.8 ml aliquots, as described previously in Scott et al. (1988), and the lavage fluid of each group of mice was pooled. Raw lavage fluid from the animals in each group was pooled to obtain concentrations of phospholipids and proteins from BAL fluid from young mouse lungs sufficient for running the assays and subsequent chemical analyses (see Oulton et al., 1999). Aliquots of each pooled raw lavage sample (henceforth referred to as RL and comprising newly released surface active alveolar surfactant and light metabolically used nonsurface active alveolar surfactant forms) were removed for phospholipid analysis, as previously described in Oulton et al. (1986) and Mason et al. (1998), and protein analysis according to the method of Lowry et al. (1951). The remainder was centrifuged at 4°C at 140g for 5 min to remove cells and debris and subsequently centrifuged at 10,000g for 30 min to pellet the heavy aggregate, newly released surface active alveolar surfactant (H) (referred to as P10 in Mason et al., 1998). The H pellet was resuspended in 1.0 ml of 0.9% NaCl and, after removing 10% aliquots for phospholipid and protein analyses, the remainder was stored at 4°C until used for surface area cycling within 24 h. For comparative purposes, the supernatant obtained from the isolation of H from each treatment group was saved for phospholipid and protein analysis. This supernatant fraction, which we will subsequently refer to as Lvivo, contains intermediate and light aggregate forms of pulmonary alveolar surfactant, together with any soluble, nonsurfactant components present at the time of killing the mice (Hawgood, 1997). In our previous studies (Mason et al., 1998; Sumarah et al., 1999), L vivo was separated into its constituent subfractions P60, P100, and S100 to determine the effect of S. chartarum and toxin on normal metabolic progression through these forms. In the present study Lvivo was not separated, as the main goal was to determine the effects of S. chartarum spores on the activity of convertase enzyme, which is responsible for converting H to Lvivo. Isolation of lamellar bodies. The postlavaged lung tissue from each mouse was surgically removed from the chest cavity. The lungs of four to five mice were pooled into the same groups as the lavage fluid analyses. The lung tissue was processed to isolate the LB according to the procedure described by Oulton et al. (1993). Briefly, this method involved the preparation of a 10% tissue homogenate in 0.01 M Tris buffer containing 0.145 M NaCl and 0.001 M EDTA (pH 7.4). The homogenate was then centrifuged at 140g to remove cells and nuclear debris. The resultant supernatant was then centrifuged at 10,000g to pellet the lamellar bodies and mitochondria, which were then resuspended in Tris buffer. Two-milliliter aliquots of the suspension were layered over discontinuous density gradients comprising 5.0 ml each of 0.68 M and 0.25 M sucrose (prepared in the same Tris buffer) and then centrifuged for 60 min at 66,000g. The band that formed between the sucrose layers (containing lamellar bodies) was removed carefully, washed by suspending in 0.9% NaCl, and centrifuged at 10,000g for 30 min. The resulting pellet, containing a highly purified preparation of lamellar bodies, was then suspended in 1.0 ml of 0.9% NaCl. Ten percent aliquots were removed from this suspension for phospholipid and protein analyses using the methods mentioned above. The remaining LB suspension was stored at 4°C and used for surface area cycling within 24 h. In vitro surface area cycling. Aliquots of H or LB, each containing 6 g of phospholipid phosphorus (Oulton et al., 1999), were made up to 1.5 ml with cycling buffer (0.15 M sodium chloride, 10 mM Tris, 1 mM calcium chloride, 1 mM magnesium chloride, and 0.1 mM EDTA (pH 7.4)) in 12 ⫻ 75 mm polypropylene tubes (Fisher Scientific, Canada). The tubes were stoppered with polypropylene plugs and mixed briefly by inversion. Surface area cycling was then performed for 3 h using the method described by Gross and Narine (1989), Veldhuizen et al. (1994), and Oulton et al. (1999). Briefly, the tubes were attached to a Rototorque rotator disk (Cole-Parmer, Chicago, IL) in an incubator at 37°C. The tubes were rotated for 3 h at 40 rotations/min so that the surface area of the content changed from 1.1 to 9 cm 2 twice each cycle. Control samples, containing the same quantity of H or LB, were maintained without cycling at 37°C. After 3 h, the contents of the control and cycled tubes were centrifuged at 10,000g for 30 min. The supernatant containing L vivo was
FIG. 1. Percent conversion of H and LB to L from untreated, saline-, C. cladosporioides-, isosatratoxin F-, and S. chartarum-exposed mice. Each sample contained 6 g of phospholipid phosphorous and was cycled at 37°C for 3 h. *Treatment groups whose average is significantly ( p ⱕ 0.05) different from the untreated control groups. **Treatment groups whose average is significantly ( p ⱕ 0.05) different from all other animal groups.
removed, made up to 2.0 ml with 0.9% NaCl, extracted with chloroform: methanol (2:1 vol/vol), and the phospholipid content was determined as described by Oulton et al. (1986). The 10,000g pellet, containing the unconverted H or LB, was resuspended in 2.0 ml of 0.9% NaCl, extracted with chloroform:methanol (2:1 vol/vol), and the phospholipid concentration was determined as described above. The percent conversion of H to L vivo and LB to L vivo was determined for each cycling tube, and the overall conversion for each sample was determined by calculating the difference in percent conversion between the cycled tubes and the uncycled, control tubes. Data analysis. The differences in percent conversion between the cycled and uncycled tubes for the untreated control and all treatment groups were first transformed to the arcsine of their square root in accordance with statistical theory (Zar, 1974). The mean ⫾ SD for the transformed percent conversion data for H and LB from untreated control mice, saline-, C. cladosporioides-, isosatratoxin F-, and S. chartarum-exposed mice were statistically compared using one-way ANOVA and Tukey’s Multiple Comparison test. Comparisons between H to L and LB to L vivo conversions for each treatment group were made using the Student’s t test. Comparisons of the protein and phospholipid concentrations RL, H, L vivo, and LB fractions were performed on untransformed data using ANOVA and Tukey’s Multiple Comparison test. Correlation coefficients for percent conversions and protein concentrations of the fractions were calculated to determine whether there was any correlation between these two variables. All tests were carried out using Systat version 5.1 and were considered significant at the 0.05 probability level.
RESULTS
Mice exposed to S. chartarum, C. cladosporioides, isosatratoxin F, or saline did not show any apparent clinical signs of respiratory distress or sickness. In Vitro Conversion of H to L vivo Figure 1 reveals that results of H to L vivo conversion in pulmonary alveolar surfactant from saline- (48.2 ⫾ 6.1%), C.
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cladosporioides- (40.9 ⫾ 7.8%), or isosatratoxin F-exposed mice (39.1 ⫾ 6.0%) were not statistically different from untreated controls (45.3 ⫾ 7.8%). Conversion from H to L vivo in the S. chartarum-exposed mice (64.5 ⫾ 7.8%) was significantly elevated ( p ⬍ 0.001) compared to all other treatment groups. In Vitro Conversion of LB to L vivo Figure 1 also reveals that in vitro conversions of LB to L vivo were not significantly different in untreated (65.8 ⫾ 7.8%) and saline-exposed (59.4 ⫾ 3.4%) mice. Conversions of LB to L vivo in LB isolated from C. cladosporioides-, isosatrotoxin F- and S. chartarum-exposed mice were all significantly depressed ( p ⬍ 0.003) compared to the LB to L vivo conversion values obtained from the untreated and saline-exposed mice. The greatest decrease in percent conversion of LB to L vivo occurred in isosatratoxin F-treated groups (42.9 ⫾ 5.2%), but this result was not significantly different from conversions in the C. cladosporioides- (44.6 ⫾ 2.6%) and S. chartarum sporetreated animals (48.3 ⫾ 8.1%). LB to L vivo conversions were significantly greater than the H to L vivo conversions in the untreated controls, saline-exposed, C. cladosporioides, and isosatratoxin F treatment groups ( p ⱕ 0.04). However, LB to L vivo was significantly less than that for H to L vivo conversion in the S. chartarum-exposed groups ( p ⬍ 0.043). Protein and Phospholipid Concentrations The protein and phospholipid concentrations of RL, H, L vivo, and LB from each treatment group are illustrated in Figs. 2A–D respectively. In each RL, H, L vivo, and LB fraction isolated from mice exposed to S. chartarum spores, the protein concentrations were significantly elevated ( p ⬍ 0.001) compared to those from untreated controls and saline- and isosatratoxin F-treated animals. The protein concentrations in the RL samples taken from mice exposed to S. chartarum averaged 6199.6 ⫾ 152.4 g/g lung, while those in the H, L vivo, and LB subfractions were 814.2 ⫾ 152.6, 624.7 ⫾ 264.3, and 767.9 ⫾ 128.3 g/g lung, respectively. The only other significant changes in protein concentration occurred in H (Fig. 2B) isolated from C. cladosporioides-exposed mice, where the protein concentration of this fraction was significantly elevated ( p ⬍ 0.001) compared to untreated and saline-treated control levels. For each fraction studied, there were no significant differences in the phospholipid concentrations between the untreated and saline-treated animal groups. In mice exposed to S. chartarum, the phospholipid concentrations of H (Fig. 2B) and LB (Fig. 2D) were significantly higher ( p ⬍ 0.005) than the saline-treated and untreated control groups, with concentrations of 813.1 ⫾ 88.4 and 1343.3 ⫾ 158.7 g/g lung, respectively, while, in L vivo, (Fig. 2C), the mean phospholipid concentration was significantly lower ( p ⬍ 0.005) at 572.6 ⫾
60.8 g/g lung. In C. cladosporioides-exposed mice, the average phospholipid concentration of L vivo was significantly lower ( p ⬍ 0.004) than the saline and untreated control groups, with a concentration of 563.3 ⫾ 91.8 g/g lung, while that of LB was significantly elevated ( p ⬍ 0.001), at a concentration of 1144.4 ⫾ 263.1 g/g lung. The only change observed in the isosatratoxin F-exposed mice was a significant elevation ( p ⬍ 0.004) in LB, with a concentration of 923.4 ⫾ 158.7 g/g lung (Fig. 2D). The correlation coefficients for H to L vivo conversion and RL and L vivo protein levels were all low (ⱕ0.2) or negative, with the exception of S. chartarum-treated mice (r ⫽ 0.611 and r ⫽ 0.304, respectively), revealing substantial positive correlation between these variables. Table 1 shows the phospholipid-to-protein ratios of the surfactant subfractions and lamellar bodies obtained from the different treatment groups. The phospholipid-to-protein ratio in LB from all treatment groups was similar. However, the phospholipid-to-protein ratios were significantly depressed ( p ⬍ 0.05) in RL, H, and L vivo from S. chartarum-treated animals, in H from C. cladosporioides-treated animals, and in L vivo from isosatratoxin F-treated mice. DISCUSSION
Our previous studies (Mason et al., 1998; Sumarah et al., 1999) have shown that intratracheal exposure to S. chartarum spores results in significant changes in the phospholipid composition and distribution in the intracellular (lamellar body) and extracellular surfactant pools. The patterns of changes in alveolar surfactant phospholipid content that we observed in these previous studies were similar to those reported by Oulton et al. (1994) and Lewis et al. (1990) in their models of lung injury, suggesting that they may be a relatively common manifestation of lung perturbation due to exposure to harmful substances. Mason et al. (1998) proposed two different mechanisms to explain the patterns of change in phospholipid content in alveolar surfactant subfractions isolated from S. chartarumexposed mice. First, they suggested that increase in the newly secreted, surface-active, subfraction (H) could be a defense reflex to rid alveolar surfaces of spores impacting on them. This type of reaction has been observed after exposure to a variety of other inhaled particles (Curti and Genghini, 1989; Jarstrand, 1984; Schurch et al., 1990). However, because this mechanism does not explain why increased alveolar surfactant secretion was not an outcome when C. cladosporioides spores stimulated mouse lung surfaces, we recently reevaluated this issue using transmission electron microscopy and stereology (Rand et al., 2001). The results of these recent studies indicate that increased surfactant production might also be a consequence of alveolar type II cell trauma associated with exposure to S. chartarum spores but not C. cladosporioides spores. Second, Mason et al. (1998) proposed that the accumulation
Stachybotrys chartarum AND ALVEOLAR SURFACTANT
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FIG. 2. Protein and phospholipid concentrations (g/g lung) in (A) RL, (B) H, (C) L vivo, and (D) LB, of untreated control, saline-, C. cladosporioides-, isosatratoxin F-, and S. chartarum-exposed mice. *Treatment groups whose average is significantly ( p ⱕ 0.05) different from the untreated and saline-treated control groups. **Treatment groups whose average is significantly ( p ⱕ 0.05) different from all other animal groups.
of the light, metabolically used pulmonary surfactant subfraction might either be due to an accelerated metabolic change of surfactant from the heavy, newly secreted (H) form to the lighter forms (L vivo) or to disruption in their clearance from the alveolar surfaces by pulmonary alveolar macrophages. Results of the present study support the hypothesis that the significant increases in light alveolar surfactant pools are a consequence of altered intraalveolar metabolism of surfactant due to a perturbation by S. chartarum spores of the convertase enzyme. As surfactant promotes lung stability by reducing the surface tension of the air–alveolar interface (Guyton et al., 1984; Notter, 1984) and performs important functions in alveolar defense (Curti and Genghini, 1989; Jarstrand, 1984; Schurch et al., 1990), altered surfactant metabolism due to exposure to S. chartarum spores may lead to lung dysfunction through diminished alveolar surfactant surface tension at-
tributes and subsequent lung stability loss and depressed pulmonary defense functions in exposed humans and animals. Our results indicate significantly depressed convertase activity in LB in the C. cladosporioides, isosatratoxin F, and S. chartarum treatment animals compared to that in the saline and untreated control groups. Because this effect does not appear to be a specific sign of S. chartarum spore- or toxin-induced lung injury, it may be a normal lung reaction to stimulation. The absence of a difference between effects of the S. chartarum and C. cladosporioides spores might mean that all fungal spores do this, while the response from the trichothecene might be for another, currently unresolved, reason. Our results also showed significantly increased in vitro H to L vivo conversion in S. chartarum-treated mice. This suggests that exposure to spores of this species may change the metabolism of extracellular surfactant through the convertase path-
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TABLE 1 Phospholipid-Protein Ratios of Alveolar Surfactant Subfractions and Lamellar Bodies Fraction Treatment
N
RL
H
L vivo
LB
Untreated control Saline Cladosporium cladosporioides Isosatratoxin F Stachybotrys chartarum
5 5 5 5 5
1.0 ⫾ 0.2 1.3 ⫾ 0.4 0.62 ⫾ 0.25 0.9 ⫾ 0.3 0.3 ⫾ 0.1**
2.4 ⫾ 0.4 2.2 ⫾ 0.7 0.8 ⫾ 0.2* 2.2 ⫾ 0.6 1.0 ⫾ 0.1*
0.63 ⫾ 0.21 0.7 ⫾ 0.19 0.28 ⫾ 0.09 0.13 ⫾ 0.32* 0.11 ⫾ 0.06*
1.4 ⫾ 0.1 2.2 ⫾ 0.7 2.2 ⫾ 0.7 1.9 ⫾ 0.1 1.8 ⫾ 0.2
Note. Mice were killed 24 h after intratracheal exposure to S. chartarum, isosatratoxin F, C. cladosporioides, or saline. Phospholipid and protein analyses were performed on aliquots of RL, H, L vivo, and LB. N ⫽ number of groups of animals used for each assay. Each value represents the phospholipid-to-protein ratio of the mean ⫾ SD for each treatment group. * Significantly different from the untreated and saline control groups ( p ⬍ 0.05). ** Significantly different from all the other control and treatment groups ( p ⬍ 0.05).
way. Increased in vitro conversion of H to L vivo in S. chartarum-treated mice is a finding that supports our in vivo study results in which we observed accumulation of the light, metabolically used pulmonary surfactant subfraction (see Mason et al., 1998). Why increased conversion was an outcome of this study is unclear. However, it may be related to the significantly increased protein content in surfactant H, which in turn could be related to the increased protein levels in RL and L vivo recovered from these animals. Protein-phospholipid ratios in these fractions from the S. chartarum-treated mice, with the exception of the lamellar bodies, showed more or equal amounts of protein to phospholipid (Table 1). A normal ratio, as was expressed in our untreated mice, is generally the opposite, more phospholipid than protein (Oulton et al., 1993; Lewis et al., 1990). Correlation coefficients for H to L vivo conversion and RL and L vivo protein levels for the S. chartarum-treated mice were also positive. As exposure to S. chartarum spores can result in severe pulmonary inflammation in mice (Nikulin et al., 1996, 1997), a likely source of the protein is from the inflammation reaction. Increased protein levels in RL and surfactant subfractions isolated from S. chartarum- and C. cladosporioidesexposed mice but not from the other treatment groups suggest that another likely consequence of spore impaction on lung tissues is serum exudation into the alveoli. Increased protein content (albumen) in BAL fluid has also been reported from rats intratracheally exposed to S. chartarum spores (Rao et al., 2000), suggesting that this response may be a common reflection of exposure to this species in different animal species. It is well documented that one of the physiological consequences of some types of lung injury involving an inflammatory response includes increases of serum fluid and soluble protein levels in the alveolar space (Lopez and Yong, 1986; Rhoades and Pflanzer, 1996; Ueda et al., 1994). Several studies have attempted to characterize the relationship between proteins, both non-surfactant-associated and surfactant-associated proteins, and pulmonary surfactant. Some of
these studies involving non-surfactant-associated proteins have documented the effect of increased alveolar protein levels on surfactant metabolism and support results of our present study. Ueda et al. (1994) found that, when isolated serum proteins were added to normal heavy subfraction surfactant (H) and cycled in vitro, accelerated H to L vivo conversion resulted. Cockshutt et al. (1990) also reported similar changes in surfactant cycling with the addition of fibrinogen to in vitro cycling surfactant. Moreover, these workers also found that increased fibrinogen also led to severe reduction of surfactant surface properties. The latter study may be relevant in S. chartarum-related inhalation exposures, especially where exposures result in lung damage in infants (Dearborne and Infeld, 1994) or animals (see Nikulin et al., 1997). As fibrinogen is a plasma protein essential for blood clotting, and as severe S. chartarum exposures can result in hemorrhagic exudation in alveoli (Nikulin et al., 1997), it is not unreasonable to expect higher concentrations of fibrinogen in alveoli, which in turn would affect surfactant metabolism and surface tension properties. Among the surfactant-associated proteins, surfactant protein (SP)-A, which is the most prevalent of the surfactant proteins, is important for maintaining the integrity of surfactant large aggregate forms (Veldhuizen et al., 1994, 1996). At least two studies have reported decreased levels of SP-A in lavage fluid taken from injured lungs (Lewis et al., 1994; Veldhuizen et al., 1993). However, the reported effects of SP-A on convertase activity are not clarified. Veldhuizen et al. (1994, 1996) suggest that decreased levels of SP-A could promote increased conversion of the heavy to the light surfactant fractions, and Cockshutt et al. (1990) observed that surfactant surface properties inhibited by fibrinogen could be restored by the addition of SP-A. We did not explore the possibility that the surface activity of surfactant was altered in S. chartarum-treated mice nor did we evaluate RL and surfactant subfractions for changes in concentrations in specific surfactant proteins such as SP-A. However, as the increased H to L vivo conversion rate observed
Stachybotrys chartarum AND ALVEOLAR SURFACTANT
in our experiments could also be related to a decreased amount of SP-A, further study is warranted to explore this possibility. ACKNOWLEDGMENTS We thank Dr. Bruce Jarvis for the gift of isosatratoxin F. This work was supported by an NSERC operating grant (T.G.R.) and a grant from the Nova Scotia Lung Association (M.O.).
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tant subtypes is altered in alveolar surfactant isolated from injured lungs. Am. Rev. Respir. Dis. 145, 1416 –1420. Hodgson, M. J., Morey, P., Leung, W.-Y., Morrow, L., Miller, D., Jarvis, B. B., Robbins, H., Halsey, J. F., and Storey, E. (1998). Building-associated pulmonary disease from exposure to Stachybotrys chartarum and Aspergillus versicolor. JOEM 40, 241–249. Jarstrand, C. (1984). Role of surfactant in the pulmonary defense system. In Pulmonary Surfactant (B. Robertson, L. M. G. van Golde, and J. J. Batenburg, Eds.). Elsevier, Amsterdam.
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Anonymous (1989). Biological Markers in Pulmonary Toxicology. National Academy Press, Washington, D.C. Brain, J. D., Knudson, D. E., Sorokin, S. P., and Davis, M. A. (1976). Pulmonary distribution of particles given by intratracheal instillation or by aerosol inhalation. Environ. Res. 11, 13–33. Burkitt, H. G., Young, B., and Heath, J. W. (1993). Wheater’s Functional Histology—A Text and Color Atlas. Churchill-Livingstone, New York. CCAC (1993). Guide to the Care and Use of Experimental Animals. Vol. 1. Bradda Printing Services, Ottawa. Cockshutt, A. M., Weitz, J., and Possmayer, F. (1990). Pulmonary surfactantassociated protein A enhances the surface activity of lipid extract surfactant and reverses inhibition by blood proteins in vitro. Biochemistry 29, 8424 – 8429. Croft, W. A., Jarvis, B. B., and Yatawara, C. S. (1986). Airborne outbreak of trichothecene toxicosis. Atmos. Environ. 30, 549 –552. Curti, P. C., and Genghini, M. (1989). Role of surfactant in alveolar defense against inhaled particles. Respiration 55(Suppl. 1), 60 – 67. Das, R. M., Ahmed, M. K., Oulton, M. R., Mantsch, H. H., Tsubai, T., and Scott, J. E. (1997). Methylmercury-induced alterations in lung and pulmonary surfactant properties of adult mice. Chem. Phys. Lipids 89, 107–11. Dearborne, D. G., and Infeld, M. D. (1994). MMWR 43, 881– 882. Dhand, R., Reyes, C., and Gross, N. J. (1994). Localization of surfactant convertase in type II pneumocytes in mice. Am. J. Respir. Crit. Care Med. 153, A92. [Abstract] Dhand, R., Teng, A., and Gross, N. J. (1996). Convertase activity in lamellar bodies. Am. J. Respir. Crit. Care Med. 153, A105. [Abstract] Gross, N. J., and Narine, K. R. (1989). Surfactant subtypes of mice: Metabolic relationships and conversion in vitro. J. Appl. Physiol. 67, 414 – 421. Gross, N. J., and Schultz, R. M. (1990). Serine protease requirements for the extracellular metabolism of pulmonary surfactant. Biochim. Biophys. Acta 1044, 222–230. Gross, N. J. (1991). Inhibition of surfactant subtype convertase in radiation model of adult respiratory distress syndrome. Am. J. Physiol. 260 (Lung Cell. Mol. Physiol. 4), L311–L317. Gross, N. J., and Schultz, R. M. (1992). Requirements for extracellular metabolism of pulmonary surfactant: Tentative identification of serine protease. Am. J. Physiol. 262, L446 –L453. Gross, N. J., Veldhuizen, R., Possmayer, F., and Dhand, R. (1997). Surfactant convertase action is not essential for surfactant film formation. Am. J. Physiol. 273 (Lung Cell. Mol. Physiol. 17), L907–L912. Guyton, A. C., Moffat, D. S., and Adair, T. H. (1984). Role of alveolar surface tension in transepithelial movement of fluid. In Pulmonary Surfactant (B. Robertson, L. M. G. van Golde, and J. J. Batenburg, Eds.), pp. 171–185. Elsevier, Amsterdam. Hawgood, S. (1997). Surfactant: Composition, structure, and metabolism. In The Lung: Scientific Foundations (R. G. Crystal, J. B. West, P. G. Barnes, and E. R. Weibel, Eds.), pp. 557–571. Lippincott-Raven Publishers, New York. Higuchi, R., Lewis, J., and Ikegami, M. (1992). In vitro conversion of surfac-
Johanning, E., Morey, P., and Jarvis, B. B. (1993). Clinical-epidemilogical investigation of health effects caused by Stachybotrys atra building contamination. Proceedings of Indoor Air 1993. Helsinki, Finland. Johanning, E., Biagini, R., Hull, D., Morey, P., Jarvis, B., and Landsbergis, P. (1996). Health and immunology study following exposure to toxigenic fungi (Stachybotrys chartarum) in a water-damaged office environment. Int. Arch. Occup. Environ. Health 68, 207–218. Lewis, J. F., Ikegami, M., and Jobe, A. H. (1990). Altered surfactant function and metabolism in rabbits with acute lung injury. J. Appl. Physiol. 69, 2303–2310. Lewis, J. F., Veldhuizen, R., Possmayer, F., Sibbald, W., Whitsett, J., Qanbar, R., and McCaig, L. (1994). Altered alveolar surfactant is a marker of acute lung injury in septic adult sheep. Am. J. Respir. Crit. Care Med. 150, 123–130. Lopez, A., and Yong, S. (1986). Injury versus inflammatory response in the lungs of rats intratracheally inoculated with bacterial lipopolysaccharide. Am. J. Vet. Res. 47, 1287–1292. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275. MaCrae, K. C., Rand, T. G., Shaw, R. A., Mason, C., Oulton, M. R., Hastings, C., Cherlet, T., Thliveris, J. A., Mantsch, H. H., MacDonald, J., and Scott, J. E. (2001). Analysis of pulmonary surfactant by Fourier-transform infrared spectroscopy following exposure to Stachybotrys chartarum (atra) spores. Chem. Phys. Lipids 110, 1–10. Mason, C. D., Rand, T. G., Oulton, M., MacDonald, J. M., and Scott, J. E. (1998). Effects of Stachybotrys chartarum (atra) conidia and isolated toxin on lung surfactant production and homeostasis. Nat. Toxins 6, 27–33. Nikulin, M., Reijula, K., Jarvis, B. B., and Hintikka, E.-L. (1996). Experimental lung mycotoxicosis in mice induced by Stachybotrys atra. Int. J. Exp. Pathol. 77, 213–218. Nikulin, M., Reijula, K., Jarvis, B., Veijalainen, P., and Hintikka, E.-L. (1997). Effects of intranasal exposure to Stachybotrys atra. Fundam. Appl. Toxicol. 35, 182–188. Notter, R. H. (1984). Surface chemistry of pulmonary surfactant: The role of the individual components. In Pulmonary Surfactant (B. Robertson, L. M. G. van Golde, and J. J. Batenburg, Eds.), pp. 17– 65. Elsevier, Amsterdam. Oulton, M., Fraser, M., Dolphin, M., Yoon, R., and Faulkner, G. (1986). Quantification of surfactant pool sizes in rabbit lung during perinatal development. J. Lipid Res. 27, 602– 612. Oulton, M., Moores, H. K., Scott, J. E., Janigan, D. T., and Hajela, R. (1991). Effects of smoke inhalation on surfactant phospholipids and phospholipase A 2 activity in mouse lung. Am. J. Pathol. 138, 195–202. Oulton, M., MacDonald, J. M., Janigan, D. T., and Faulkner, G. T. (1993). Mouse alveolar subfractions: Characterization of subtypes prepared by differential centrifugation. Lipids 28, 715–720. Oulton, M. R., Janigan, D. T., MacDonald, J. M. R., Faulkner, G. T., and Scott, J. E. (1994). Effects of smoke inhalation on alveolar surfactant subtypes in mice. Am. J. Pathol. 145, 941–950.
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Oulton, M., Edwards, E., and Handa, K. (1999). Convertase activity in alveolar surfactant and lamellar bodies in fetal, newborn and adult rabbits. J. Appl. Physiol. 86, 71–77. Rand, T. G., Mahoney, M., White, K., and Oulton, M. (2001). Micro-anatomical changes associated with alveolar type II cells in juvenile mice exposed to Stachybotrys chartarum and isolated toxin. (Submitted for publication). Rao, C. Y., Brain, J. D., and Burge, H. A. (2000). Reduction of pulmonary toxicity of Stachybotrys chartarum spores by methanol extraction of mycotoxins. Appl. Environ. Microbiol. 66, 2817–2821. Rhoades, R., and Pflanzer, R. (1996). Human Physiology. Saunders College Publishing, Toronto. Riley, R. T., and Norred, W. P. (1996). Mechanisms of mycotoxicity. In The Mycota VI (Howard, D. H., and J. D. Miller, Eds.). Springer-Verlag, Berlin. Schurch, S., Gehr, P., Hof, V. I., Geiser, M., and Green, F. (1990). Surfactant displaces particles toward the epithelium in airways and alveoli. Respir. Physiol. 80, 17–32. Scott, J. E., Possmayer, F., Quire, M. A., Tanswell, A. K., and Harding, P. G. R. (1986). Alveolar pre type II cells from the fetal rabbit lung: Characterization of the production of disaturated phospholipid during cellular differentiation. Biochim. Biophys. Acta 879, 292–300.
(1987). Trichothecene mycotoxins in aerosolized conidia of Stachybotrys atra. Appl. Environ. Microbiol. 53, 1370 –1375. Sumarah, M. W., Rand, T. G., Oulton, M., Mason, C. D., MacDonald, J., and Anthes, M. (1999). Effects of Stachybotrys chartarum spores and toxin on alveolar surfactant phospholipid composition and concentration in mice. Proceedings of the 3rd International Conference on Bioaerosols, Fungi and Mycotoxins: Health Effects, Assessment, Prevention and Control. Saratoga Springs, NY. Ueda, T., Ikegami, M., and Jobe, A. (1994). Surfactant subtypes: In vitro conversion, in vivo function, and effects of serum proteins. Am. J. Respir. Crit. Care Med. 149, 1254 –1259. Veldhuizen, R. A., Lee, J., Sandler, D., Hull, W., Whitsett, J. A., Lewis, J., Possmayer, F., and Norvik, R. J. (1993). Alterations in pulmonary surfactant composition and activity after experimental lung transplantation. Am. Rev. Respir. Dis. 148, 208 –215. Veldhuizen, A. W., Hearn, S. A., Lewis, J. F., and Possmayer, F. (1994). Surface-area cycling of different surfactant preparations: SP-A and SP-B are essential for large-aggregate integrity. Biochem. J. 300, 519 –524. Veldhuizen, R., Yao, L., Hearn, S., Possmayer, F., and Lewis, J. (1996). Surfactant-associated protein A is important for maintaining surfactant large-aggregate forms during surface-area cycling. Biochem. J. 313, 835– 840.
Scott, J. E., Forkett, P. G., Oulton, M., Rasmusson, M. G., Temple, S., Fraser, M. O., and Whitfield, S. (1988). Pulmonary toxicity of trichloroethylene: Introduction of changes in surfactant phospholipids and phospholipase A 2 activity in the mouse lung. Exp. Mol. Pathol. 49, 141–150.
Veldhuizen, R. A. W., Ito, Y., Marcou, J., Yao, L.-J., McCaig, L., and Lewis, J. F. (1997). Effects of lung injury on pulmonary surfactant aggregate conversion in vivo and in vitro. Am. J. Physiol. 272 (Lung Cell. Mol. Physiol. 161) L872–L878.
Sorenson, W. G., Frazier, D. G., Jarvis, B. B., Simpson, J., and Robinson, V.
Zar, J. H. (1974). Biostatistical Analysis. Prentice-Hall, New Jersey.
Effects of Stachybotrys chartarum on Surfactant Convertase Activity in Juvenile Mice C. D. Mason,* T. G. Rand,* M. Oulton,† J. MacDonald,† and M. Anthes† *Department of Biology, Saint Mary’s University, Halifax, Nova Scotia, Canada; and †Department of Physiology and Obstetrics and Gynecology, Dalhousie University, Halifax, Nova Scotia, Canada August 2, 2000; accepted January 9, 2001
nificantly elevated compared to those from other treatment groups, while LB phospholipid concentrations were significantly increased compared to saline and untreated control animal groups. These results show that S. chartarum spores significantly alter convertase activity in both the H and LB surfactant fractions in juvenile mice and that these changes can be related to changes in protein and phospholipid concentrations in alveolar lavage fractions. As surfactant promotes lung stability by reducing the surface tension of the air–alveolar interface, these results further support our position that inhalation exposure to S. chartarum spores in exposed individuals may lead to altered surfactant metabolism, and possibly to lung dysfunction through diminished alveolar surfactant surface tension attributes, and lung stability. © 2001 Academic Press Key Words: alveolar type II cells; convertase; fungal conidia; satratoxins; intratracheal instillation; surfactant.
Effects of Stachybotrys chartarum on Surfactant Convertase Activity in Juvenile Mice. Mason, C. D., Rand, T. G., Oulton, M., MacDonald, J., and Anthes, M. (2001). Toxicol. Appl. Pharmacol. 172, 21–28. We have shown recently that alveolar type II cells are sensitive to exposure to Stachybotrys chartarum spores, both in vitro and in an in vivo juvenile mouse model. In mice, this sensitivity is manifest in part as a significant increase in the newly secreted, biologically active, heavy aggregate form of alveolar surfactant (H) and the accumulation of the lighter, “metabolically used”, biologically inactive alveolar surfactant forms (L vivo) in the interalveolar space. Conversion of the heavy, surface-active alveolar surfactant to the light metabolically used, nonsurface active forms is believed to involve the activity of an enzyme, namely convertase, which is thought to be derived from lamellar bodies (LB) in alveolar type II cells. The purpose of this study was to evaluate the effects of S. chartarum spores on mouse H and LB convertase activity by measuring their rates of conversion to L vivo using the in vitro surface area cycling technique. It was determined whether there were concurrent changes in the protein and phospholipid concentrations of the raw bronchoalveolar lavage fluid (RL) and LB fractions that could be correlated with changes in convertase activity. Conversions of H to L vivo in untreated control mice and saline-, isosatratoxin F-, and Cladosporium cladosporioides-exposed mice were not significantly different (p > 0.05). However, conversion from H to L vivo in the mice exposed to S. chartarum spores was significantly higher than all other treatment groups (p < 0.001). LB to L vivo conversions in untreated and salineexposed mice were not significantly different, although they were significantly higher than the H to L vivo conversions in these two animal treatment groups (p < 0.005), which supports the position that LB is a source of convertase activity in animals. LB to L vivo conversion from C. cladosporioides-, isosatrotoxin F-, and S. chartarum-exposed mice were all significantly depressed (p < 0.003) compared to the LB to L vivo conversion values obtained from untreated and saline-exposed mice. Protein concentrations in RL, H, L vivo, and LB from mice exposed to S. chartarum spores were significantly elevated compared to those from the other treatment groups (p < 0.001). Protein concentration in H isolated from C. cladosporioides-exposed mice was also significantly elevated above untreated and saline control animal levels. Phospholipid concentrations in H isolated from S. chartarum-exposed mice were sig-
Stachybotrys chartarum (atra) is an important toxigenic fungus often associated with chronically wet cellulose-based building materials. Occupational inhalation exposure to the spores, mycelial fragments, and dust, all containing toxins of this species, has been implicated as the cause of a variety of health problems. Symptoms include mucous membrane irritation, respiratory and nervous system disorders, and immune system dysfunction (Croft et al., 1986; Hodgson et al., 1998; Johanning et al., 1993, 1996). Despite the variety of symptoms associated with inhalation exposure to S. chartarum, there have been only a few in vivo animal studies evaluating the impact of spores of this species on lung tissue. Studies in inhalation toxicology have demonstrated the usefulness of bronchoalveolar lavage (BAL) fluid analysis for evaluating pulmonary dysfunction and damage (Anonymous, 1989; Das et al., 1997; Lopez and Yong, 1986; Oulton et al., 1993, 1994; Veldhuizen et al., 1997). The methods used are considered highly sensitive, quantifiable, and reproducible, with numerous advantages over routine histopathological evaluation of lung (Lopez and Yong, 1986). Previous work in our laboratory has led to the development of a relatively simple but useful method for evaluating pulmonary cell damage and lung dysfunction independent of the concurrent inflammatory lung 21
0041-008X/01 $35.00 Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.
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response caused by exposure to toxic particulates (Oulton et al., 1991, 1993; Mason et al., 1998; Sumarah et al., 1999). This method is based on changes in the distribution or composition of alveolar surfactant subfractions, which are easily sampled and evaluated from the BAL fluid (Oulton et al., 1991, 1993). Mason et al. (1998) and Sumarah et al. (1999) used this methodology to show that alveolar type II cells, which comprise about 60% of the alveolar lining epithelium (Burkitt et al., 1993), are highly sensitive to exposure to S. chartarum spores. This sensitivity is manifest by significant alterations in the normal metabolic processing of alveolar surfactant in experimentally exposed mice. Mason et al. (1998) demonstrated that this sensitivity involves a significant increase in the newly secreted, biologically active, heavy aggregate form of alveolar surfactant and the subsequent accumulation of the lighter, “metabolically used”, biologically inactive alveolar surfactant forms in the interalveolar space. Sumarah et al. (1999) have shown that S. chartarum spore and toxin exposure induces significant changes in the phospholipid composition in the in vivo mouse model of lung injury, including depression of disaturated phosphatidylcholine (DSPC), the major surfactant phospholipid, which we believe may lead to lung dysfunction through altered pulmonary surfactant surface tension attributes. MaCrae et al. (2001) have revealed that the pulmonary surfactant compositional changes in S. chartarum spore-exposed mice may be due to molecular and biochemical alterations of the phospholipid components of pulmonary surfactant, resulting in a significant elevation in lysophospholipid content, which is a recognized accompaniment of alveolar type II cell damage (Scott et al., 1986, 1988). Mason et al. (1998) suggested that the accumulation of the metabolically used, biologically inactive alveolar surfactant forms could be the result of a disruption in the conversion of heavy surfactant to light or in the mechanisms of clearance of the converted surfactant from the alveolar space. Conversion of the heavy to light forms of pulmonary surfactant requires the activity of a convertase enzyme, a tentatively identified 76-kDa serine protease (Gross and Schultz, 1990, 1992). This enzyme has been identified in many species, including mice (Dhand et al., 1994). Studies by Dhand et al. (1996) and Oulton et al. (1999) suggest that lamellar bodies (LB), the intracellular storage form of surfactant, may be the source of this activity. Gross and Narine (1989) developed a method to study convertase activity in vitro. The method involves an end-over-end rotation of surfactant samples, which mimics the changing surface area of the lungs during respiration. This technique has been used by investigators to characterize the activity of the convertase enzyme and alveolar surfactant metabolism (Gross and Schultz, 1992; Oulton et al., 1999; Ueda et al., 1994) and to assess its activity in lamellar bodies (Oulton et al., 1999; Dhand et al., 1996). This technique has also been used to give insight into the effects of lung injury on intraalveolar metabolism of surfactant (Gross, 1991; Higuchi et al., 1992; Ueda et al., 1994; Veldhuizen et al., 1997).
The purpose of the present study was to use the in vitro cycling technique to determine whether S. chartarum spore exposure affects the conversion of heavy to light alveolar surfactant subfractions and/or convertase activity in lamellar bodies and contributes to the accumulation of the light form isolated from lung tissues of juvenile mice. It was also to determine whether there were concurrent changes in the protein and phospholipid concentrations of the raw bronchoalveolar lavage fluid (RL) and LB fractions that could be correlated with changes in convertase activity. Based on results of our previous studies, we hypothesize that the accumulation of the metabolically used, biologically inactive alveolar surfactant forms in mouse lung is the result of a disruption in the conversion of heavy surfactant to the lighter, biologically inactive alveolar surfactant forms. METHODS Preparation of treatments. The S. chartarum isolate (spore dimensions 11.6 ⫾ 1.2 m long ⫻ 5.7 ⫾ 0.92 m wide; n ⫽ 240) used was recovered from a Hawaiian hotel and has been deposited in the Department of Agriculture Canada Culture Collection (DAOM 225489). The Cladosporium cladosporioides isolate (spore dimensions 6.3 ⫾ 0.9 m long ⫻ 2.5 ⫾ 0.51 m wide; n ⫽ 200) was used as a negative control and was recovered from an outdoor sampling site in Nova Scotia (T.G.R.). Both isolates were cultured on 2% MEA, at 25°C for 2 to 3 weeks. The spores were harvested by flooding the cultures with sterile 0.9% NaCl followed by gentle agitation using a heatsterilized Pasteur pipette, and then collected and concentrated by centrifugation at 5000 rpm for 3 min in 1.5-ml Eppendorf centrifuge tubes. The spores were then suspended in 0.9% NaCl at a concentration of 1.4 ⫻ 10 6 conidia/ml, as per methods previously described in Mason et al. (1998). The suspensions were each used for intratracheal instillations within 2 h of isolation. Isosatratoxin F was employed as a positive control to determine whether the observed effects were due to exposure to trichothecene toxin sequestered in S. chartarum spores. It was isolated from an S. chartarum strain by Dr. Bruce Jarvis of the University of Maryland (College Park, MD). The toxin was dissolved in absolute methanol and then mixed to a concentration of 10 g/ml in 0.9% NaCl. Animals. Random-bred, pathogen-free, Carworth Farms White (CFW), Swiss Webster, male mice, 3– 4 weeks old were used in these experiments. The mice were housed according to the standards of the Canadian Council for Animal Care (CCAC, 1993). The mice were given food and water ad libitum. The animals were acclimatized for 1 week prior to usage. Intratracheal instillations. Control (untreated) and treatment animals (S. chartarum, C. cladosporioides, isosatratoxin F, and saline) were separated into groups of four or five mice each. A minimum of five groups was reserved for each treatment. The mice were anesthetized for instillation using an intramuscular injection of a mixture of ketamine (Ketaleen) and xylazine (Rompun) as previously described in Mason et al. (1998). The intratracheal instillation procedure used was a modification of the method described by Brain et al. (1976). It is described in detail in Mason et al. (1998). Each mouse, with the exception of untreated control mice, was instilled with either 50 l of S. chartarum or C. cladosporioides (1.4 ⫻ 10 6 conidia/ml), 10 g/ml of isosatratoxin F, or 0.9% NaCl. The mice were put back into their cages immediately after instillation and allowed to recover for 24 h. During recovery, the mice were continuously monitored for signs of sickness or distress as outlined in CCAC guidelines (CCAC, 1993). If the animals showed signs of distress, they were immediately euthanized using a sodium pentobarbitol (65 mg/ml) overdose and excluded from the study. Isolation of heavy aggregate alveolar surfactant. The exposed mice were killed after 24 h using a 300 l ip injection of 65 mg/ml sodium pentobarbitol. This
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Stachybotrys chartarum AND ALVEOLAR SURFACTANT time was chosen because results of our previous studies (see Mason et al., 1998; Sumarah et al., 1999) indicated that the most significant changes in surfactant composition and homeostasis were manifest between 24 and 48 h postinoculation. Mouse lungs were lavaged with 0.9% NaCl in 4 ⫻ 0.8 ml aliquots, as described previously in Scott et al. (1988), and the lavage fluid of each group of mice was pooled. Raw lavage fluid from the animals in each group was pooled to obtain concentrations of phospholipids and proteins from BAL fluid from young mouse lungs sufficient for running the assays and subsequent chemical analyses (see Oulton et al., 1999). Aliquots of each pooled raw lavage sample (henceforth referred to as RL and comprising newly released surface active alveolar surfactant and light metabolically used nonsurface active alveolar surfactant forms) were removed for phospholipid analysis, as previously described in Oulton et al. (1986) and Mason et al. (1998), and protein analysis according to the method of Lowry et al. (1951). The remainder was centrifuged at 4°C at 140g for 5 min to remove cells and debris and subsequently centrifuged at 10,000g for 30 min to pellet the heavy aggregate, newly released surface active alveolar surfactant (H) (referred to as P10 in Mason et al., 1998). The H pellet was resuspended in 1.0 ml of 0.9% NaCl and, after removing 10% aliquots for phospholipid and protein analyses, the remainder was stored at 4°C until used for surface area cycling within 24 h. For comparative purposes, the supernatant obtained from the isolation of H from each treatment group was saved for phospholipid and protein analysis. This supernatant fraction, which we will subsequently refer to as Lvivo, contains intermediate and light aggregate forms of pulmonary alveolar surfactant, together with any soluble, nonsurfactant components present at the time of killing the mice (Hawgood, 1997). In our previous studies (Mason et al., 1998; Sumarah et al., 1999), L vivo was separated into its constituent subfractions P60, P100, and S100 to determine the effect of S. chartarum and toxin on normal metabolic progression through these forms. In the present study Lvivo was not separated, as the main goal was to determine the effects of S. chartarum spores on the activity of convertase enzyme, which is responsible for converting H to Lvivo. Isolation of lamellar bodies. The postlavaged lung tissue from each mouse was surgically removed from the chest cavity. The lungs of four to five mice were pooled into the same groups as the lavage fluid analyses. The lung tissue was processed to isolate the LB according to the procedure described by Oulton et al. (1993). Briefly, this method involved the preparation of a 10% tissue homogenate in 0.01 M Tris buffer containing 0.145 M NaCl and 0.001 M EDTA (pH 7.4). The homogenate was then centrifuged at 140g to remove cells and nuclear debris. The resultant supernatant was then centrifuged at 10,000g to pellet the lamellar bodies and mitochondria, which were then resuspended in Tris buffer. Two-milliliter aliquots of the suspension were layered over discontinuous density gradients comprising 5.0 ml each of 0.68 M and 0.25 M sucrose (prepared in the same Tris buffer) and then centrifuged for 60 min at 66,000g. The band that formed between the sucrose layers (containing lamellar bodies) was removed carefully, washed by suspending in 0.9% NaCl, and centrifuged at 10,000g for 30 min. The resulting pellet, containing a highly purified preparation of lamellar bodies, was then suspended in 1.0 ml of 0.9% NaCl. Ten percent aliquots were removed from this suspension for phospholipid and protein analyses using the methods mentioned above. The remaining LB suspension was stored at 4°C and used for surface area cycling within 24 h. In vitro surface area cycling. Aliquots of H or LB, each containing 6 g of phospholipid phosphorus (Oulton et al., 1999), were made up to 1.5 ml with cycling buffer (0.15 M sodium chloride, 10 mM Tris, 1 mM calcium chloride, 1 mM magnesium chloride, and 0.1 mM EDTA (pH 7.4)) in 12 ⫻ 75 mm polypropylene tubes (Fisher Scientific, Canada). The tubes were stoppered with polypropylene plugs and mixed briefly by inversion. Surface area cycling was then performed for 3 h using the method described by Gross and Narine (1989), Veldhuizen et al. (1994), and Oulton et al. (1999). Briefly, the tubes were attached to a Rototorque rotator disk (Cole-Parmer, Chicago, IL) in an incubator at 37°C. The tubes were rotated for 3 h at 40 rotations/min so that the surface area of the content changed from 1.1 to 9 cm 2 twice each cycle. Control samples, containing the same quantity of H or LB, were maintained without cycling at 37°C. After 3 h, the contents of the control and cycled tubes were centrifuged at 10,000g for 30 min. The supernatant containing L vivo was
FIG. 1. Percent conversion of H and LB to L from untreated, saline-, C. cladosporioides-, isosatratoxin F-, and S. chartarum-exposed mice. Each sample contained 6 g of phospholipid phosphorous and was cycled at 37°C for 3 h. *Treatment groups whose average is significantly ( p ⱕ 0.05) different from the untreated control groups. **Treatment groups whose average is significantly ( p ⱕ 0.05) different from all other animal groups.
removed, made up to 2.0 ml with 0.9% NaCl, extracted with chloroform: methanol (2:1 vol/vol), and the phospholipid content was determined as described by Oulton et al. (1986). The 10,000g pellet, containing the unconverted H or LB, was resuspended in 2.0 ml of 0.9% NaCl, extracted with chloroform:methanol (2:1 vol/vol), and the phospholipid concentration was determined as described above. The percent conversion of H to L vivo and LB to L vivo was determined for each cycling tube, and the overall conversion for each sample was determined by calculating the difference in percent conversion between the cycled tubes and the uncycled, control tubes. Data analysis. The differences in percent conversion between the cycled and uncycled tubes for the untreated control and all treatment groups were first transformed to the arcsine of their square root in accordance with statistical theory (Zar, 1974). The mean ⫾ SD for the transformed percent conversion data for H and LB from untreated control mice, saline-, C. cladosporioides-, isosatratoxin F-, and S. chartarum-exposed mice were statistically compared using one-way ANOVA and Tukey’s Multiple Comparison test. Comparisons between H to L and LB to L vivo conversions for each treatment group were made using the Student’s t test. Comparisons of the protein and phospholipid concentrations RL, H, L vivo, and LB fractions were performed on untransformed data using ANOVA and Tukey’s Multiple Comparison test. Correlation coefficients for percent conversions and protein concentrations of the fractions were calculated to determine whether there was any correlation between these two variables. All tests were carried out using Systat version 5.1 and were considered significant at the 0.05 probability level.
RESULTS
Mice exposed to S. chartarum, C. cladosporioides, isosatratoxin F, or saline did not show any apparent clinical signs of respiratory distress or sickness. In Vitro Conversion of H to L vivo Figure 1 reveals that results of H to L vivo conversion in pulmonary alveolar surfactant from saline- (48.2 ⫾ 6.1%), C.
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cladosporioides- (40.9 ⫾ 7.8%), or isosatratoxin F-exposed mice (39.1 ⫾ 6.0%) were not statistically different from untreated controls (45.3 ⫾ 7.8%). Conversion from H to L vivo in the S. chartarum-exposed mice (64.5 ⫾ 7.8%) was significantly elevated ( p ⬍ 0.001) compared to all other treatment groups. In Vitro Conversion of LB to L vivo Figure 1 also reveals that in vitro conversions of LB to L vivo were not significantly different in untreated (65.8 ⫾ 7.8%) and saline-exposed (59.4 ⫾ 3.4%) mice. Conversions of LB to L vivo in LB isolated from C. cladosporioides-, isosatrotoxin F- and S. chartarum-exposed mice were all significantly depressed ( p ⬍ 0.003) compared to the LB to L vivo conversion values obtained from the untreated and saline-exposed mice. The greatest decrease in percent conversion of LB to L vivo occurred in isosatratoxin F-treated groups (42.9 ⫾ 5.2%), but this result was not significantly different from conversions in the C. cladosporioides- (44.6 ⫾ 2.6%) and S. chartarum sporetreated animals (48.3 ⫾ 8.1%). LB to L vivo conversions were significantly greater than the H to L vivo conversions in the untreated controls, saline-exposed, C. cladosporioides, and isosatratoxin F treatment groups ( p ⱕ 0.04). However, LB to L vivo was significantly less than that for H to L vivo conversion in the S. chartarum-exposed groups ( p ⬍ 0.043). Protein and Phospholipid Concentrations The protein and phospholipid concentrations of RL, H, L vivo, and LB from each treatment group are illustrated in Figs. 2A–D respectively. In each RL, H, L vivo, and LB fraction isolated from mice exposed to S. chartarum spores, the protein concentrations were significantly elevated ( p ⬍ 0.001) compared to those from untreated controls and saline- and isosatratoxin F-treated animals. The protein concentrations in the RL samples taken from mice exposed to S. chartarum averaged 6199.6 ⫾ 152.4 g/g lung, while those in the H, L vivo, and LB subfractions were 814.2 ⫾ 152.6, 624.7 ⫾ 264.3, and 767.9 ⫾ 128.3 g/g lung, respectively. The only other significant changes in protein concentration occurred in H (Fig. 2B) isolated from C. cladosporioides-exposed mice, where the protein concentration of this fraction was significantly elevated ( p ⬍ 0.001) compared to untreated and saline-treated control levels. For each fraction studied, there were no significant differences in the phospholipid concentrations between the untreated and saline-treated animal groups. In mice exposed to S. chartarum, the phospholipid concentrations of H (Fig. 2B) and LB (Fig. 2D) were significantly higher ( p ⬍ 0.005) than the saline-treated and untreated control groups, with concentrations of 813.1 ⫾ 88.4 and 1343.3 ⫾ 158.7 g/g lung, respectively, while, in L vivo, (Fig. 2C), the mean phospholipid concentration was significantly lower ( p ⬍ 0.005) at 572.6 ⫾
60.8 g/g lung. In C. cladosporioides-exposed mice, the average phospholipid concentration of L vivo was significantly lower ( p ⬍ 0.004) than the saline and untreated control groups, with a concentration of 563.3 ⫾ 91.8 g/g lung, while that of LB was significantly elevated ( p ⬍ 0.001), at a concentration of 1144.4 ⫾ 263.1 g/g lung. The only change observed in the isosatratoxin F-exposed mice was a significant elevation ( p ⬍ 0.004) in LB, with a concentration of 923.4 ⫾ 158.7 g/g lung (Fig. 2D). The correlation coefficients for H to L vivo conversion and RL and L vivo protein levels were all low (ⱕ0.2) or negative, with the exception of S. chartarum-treated mice (r ⫽ 0.611 and r ⫽ 0.304, respectively), revealing substantial positive correlation between these variables. Table 1 shows the phospholipid-to-protein ratios of the surfactant subfractions and lamellar bodies obtained from the different treatment groups. The phospholipid-to-protein ratio in LB from all treatment groups was similar. However, the phospholipid-to-protein ratios were significantly depressed ( p ⬍ 0.05) in RL, H, and L vivo from S. chartarum-treated animals, in H from C. cladosporioides-treated animals, and in L vivo from isosatratoxin F-treated mice. DISCUSSION
Our previous studies (Mason et al., 1998; Sumarah et al., 1999) have shown that intratracheal exposure to S. chartarum spores results in significant changes in the phospholipid composition and distribution in the intracellular (lamellar body) and extracellular surfactant pools. The patterns of changes in alveolar surfactant phospholipid content that we observed in these previous studies were similar to those reported by Oulton et al. (1994) and Lewis et al. (1990) in their models of lung injury, suggesting that they may be a relatively common manifestation of lung perturbation due to exposure to harmful substances. Mason et al. (1998) proposed two different mechanisms to explain the patterns of change in phospholipid content in alveolar surfactant subfractions isolated from S. chartarumexposed mice. First, they suggested that increase in the newly secreted, surface-active, subfraction (H) could be a defense reflex to rid alveolar surfaces of spores impacting on them. This type of reaction has been observed after exposure to a variety of other inhaled particles (Curti and Genghini, 1989; Jarstrand, 1984; Schurch et al., 1990). However, because this mechanism does not explain why increased alveolar surfactant secretion was not an outcome when C. cladosporioides spores stimulated mouse lung surfaces, we recently reevaluated this issue using transmission electron microscopy and stereology (Rand et al., 2001). The results of these recent studies indicate that increased surfactant production might also be a consequence of alveolar type II cell trauma associated with exposure to S. chartarum spores but not C. cladosporioides spores. Second, Mason et al. (1998) proposed that the accumulation
Stachybotrys chartarum AND ALVEOLAR SURFACTANT
25
FIG. 2. Protein and phospholipid concentrations (g/g lung) in (A) RL, (B) H, (C) L vivo, and (D) LB, of untreated control, saline-, C. cladosporioides-, isosatratoxin F-, and S. chartarum-exposed mice. *Treatment groups whose average is significantly ( p ⱕ 0.05) different from the untreated and saline-treated control groups. **Treatment groups whose average is significantly ( p ⱕ 0.05) different from all other animal groups.
of the light, metabolically used pulmonary surfactant subfraction might either be due to an accelerated metabolic change of surfactant from the heavy, newly secreted (H) form to the lighter forms (L vivo) or to disruption in their clearance from the alveolar surfaces by pulmonary alveolar macrophages. Results of the present study support the hypothesis that the significant increases in light alveolar surfactant pools are a consequence of altered intraalveolar metabolism of surfactant due to a perturbation by S. chartarum spores of the convertase enzyme. As surfactant promotes lung stability by reducing the surface tension of the air–alveolar interface (Guyton et al., 1984; Notter, 1984) and performs important functions in alveolar defense (Curti and Genghini, 1989; Jarstrand, 1984; Schurch et al., 1990), altered surfactant metabolism due to exposure to S. chartarum spores may lead to lung dysfunction through diminished alveolar surfactant surface tension at-
tributes and subsequent lung stability loss and depressed pulmonary defense functions in exposed humans and animals. Our results indicate significantly depressed convertase activity in LB in the C. cladosporioides, isosatratoxin F, and S. chartarum treatment animals compared to that in the saline and untreated control groups. Because this effect does not appear to be a specific sign of S. chartarum spore- or toxin-induced lung injury, it may be a normal lung reaction to stimulation. The absence of a difference between effects of the S. chartarum and C. cladosporioides spores might mean that all fungal spores do this, while the response from the trichothecene might be for another, currently unresolved, reason. Our results also showed significantly increased in vitro H to L vivo conversion in S. chartarum-treated mice. This suggests that exposure to spores of this species may change the metabolism of extracellular surfactant through the convertase path-
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MASON ET AL.
TABLE 1 Phospholipid-Protein Ratios of Alveolar Surfactant Subfractions and Lamellar Bodies Fraction Treatment
N
RL
H
L vivo
LB
Untreated control Saline Cladosporium cladosporioides Isosatratoxin F Stachybotrys chartarum
5 5 5 5 5
1.0 ⫾ 0.2 1.3 ⫾ 0.4 0.62 ⫾ 0.25 0.9 ⫾ 0.3 0.3 ⫾ 0.1**
2.4 ⫾ 0.4 2.2 ⫾ 0.7 0.8 ⫾ 0.2* 2.2 ⫾ 0.6 1.0 ⫾ 0.1*
0.63 ⫾ 0.21 0.7 ⫾ 0.19 0.28 ⫾ 0.09 0.13 ⫾ 0.32* 0.11 ⫾ 0.06*
1.4 ⫾ 0.1 2.2 ⫾ 0.7 2.2 ⫾ 0.7 1.9 ⫾ 0.1 1.8 ⫾ 0.2
Note. Mice were killed 24 h after intratracheal exposure to S. chartarum, isosatratoxin F, C. cladosporioides, or saline. Phospholipid and protein analyses were performed on aliquots of RL, H, L vivo, and LB. N ⫽ number of groups of animals used for each assay. Each value represents the phospholipid-to-protein ratio of the mean ⫾ SD for each treatment group. * Significantly different from the untreated and saline control groups ( p ⬍ 0.05). ** Significantly different from all the other control and treatment groups ( p ⬍ 0.05).
way. Increased in vitro conversion of H to L vivo in S. chartarum-treated mice is a finding that supports our in vivo study results in which we observed accumulation of the light, metabolically used pulmonary surfactant subfraction (see Mason et al., 1998). Why increased conversion was an outcome of this study is unclear. However, it may be related to the significantly increased protein content in surfactant H, which in turn could be related to the increased protein levels in RL and L vivo recovered from these animals. Protein-phospholipid ratios in these fractions from the S. chartarum-treated mice, with the exception of the lamellar bodies, showed more or equal amounts of protein to phospholipid (Table 1). A normal ratio, as was expressed in our untreated mice, is generally the opposite, more phospholipid than protein (Oulton et al., 1993; Lewis et al., 1990). Correlation coefficients for H to L vivo conversion and RL and L vivo protein levels for the S. chartarum-treated mice were also positive. As exposure to S. chartarum spores can result in severe pulmonary inflammation in mice (Nikulin et al., 1996, 1997), a likely source of the protein is from the inflammation reaction. Increased protein levels in RL and surfactant subfractions isolated from S. chartarum- and C. cladosporioidesexposed mice but not from the other treatment groups suggest that another likely consequence of spore impaction on lung tissues is serum exudation into the alveoli. Increased protein content (albumen) in BAL fluid has also been reported from rats intratracheally exposed to S. chartarum spores (Rao et al., 2000), suggesting that this response may be a common reflection of exposure to this species in different animal species. It is well documented that one of the physiological consequences of some types of lung injury involving an inflammatory response includes increases of serum fluid and soluble protein levels in the alveolar space (Lopez and Yong, 1986; Rhoades and Pflanzer, 1996; Ueda et al., 1994). Several studies have attempted to characterize the relationship between proteins, both non-surfactant-associated and surfactant-associated proteins, and pulmonary surfactant. Some of
these studies involving non-surfactant-associated proteins have documented the effect of increased alveolar protein levels on surfactant metabolism and support results of our present study. Ueda et al. (1994) found that, when isolated serum proteins were added to normal heavy subfraction surfactant (H) and cycled in vitro, accelerated H to L vivo conversion resulted. Cockshutt et al. (1990) also reported similar changes in surfactant cycling with the addition of fibrinogen to in vitro cycling surfactant. Moreover, these workers also found that increased fibrinogen also led to severe reduction of surfactant surface properties. The latter study may be relevant in S. chartarum-related inhalation exposures, especially where exposures result in lung damage in infants (Dearborne and Infeld, 1994) or animals (see Nikulin et al., 1997). As fibrinogen is a plasma protein essential for blood clotting, and as severe S. chartarum exposures can result in hemorrhagic exudation in alveoli (Nikulin et al., 1997), it is not unreasonable to expect higher concentrations of fibrinogen in alveoli, which in turn would affect surfactant metabolism and surface tension properties. Among the surfactant-associated proteins, surfactant protein (SP)-A, which is the most prevalent of the surfactant proteins, is important for maintaining the integrity of surfactant large aggregate forms (Veldhuizen et al., 1994, 1996). At least two studies have reported decreased levels of SP-A in lavage fluid taken from injured lungs (Lewis et al., 1994; Veldhuizen et al., 1993). However, the reported effects of SP-A on convertase activity are not clarified. Veldhuizen et al. (1994, 1996) suggest that decreased levels of SP-A could promote increased conversion of the heavy to the light surfactant fractions, and Cockshutt et al. (1990) observed that surfactant surface properties inhibited by fibrinogen could be restored by the addition of SP-A. We did not explore the possibility that the surface activity of surfactant was altered in S. chartarum-treated mice nor did we evaluate RL and surfactant subfractions for changes in concentrations in specific surfactant proteins such as SP-A. However, as the increased H to L vivo conversion rate observed
Stachybotrys chartarum AND ALVEOLAR SURFACTANT
in our experiments could also be related to a decreased amount of SP-A, further study is warranted to explore this possibility. ACKNOWLEDGMENTS We thank Dr. Bruce Jarvis for the gift of isosatratoxin F. This work was supported by an NSERC operating grant (T.G.R.) and a grant from the Nova Scotia Lung Association (M.O.).
27
tant subtypes is altered in alveolar surfactant isolated from injured lungs. Am. Rev. Respir. Dis. 145, 1416 –1420. Hodgson, M. J., Morey, P., Leung, W.-Y., Morrow, L., Miller, D., Jarvis, B. B., Robbins, H., Halsey, J. F., and Storey, E. (1998). Building-associated pulmonary disease from exposure to Stachybotrys chartarum and Aspergillus versicolor. JOEM 40, 241–249. Jarstrand, C. (1984). Role of surfactant in the pulmonary defense system. In Pulmonary Surfactant (B. Robertson, L. M. G. van Golde, and J. J. Batenburg, Eds.). Elsevier, Amsterdam.
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